Methods are known for detecting analytes in a sample with high specificity and sensitivity. These methods include antigen-antibody assays as well DNA hybridization based assays. Detection of analytes using immunodetection is well known in the art. Methods include direct labeling of antibodies using radioisotopes, fluorescent or chemiluminescent tags, or ELISA assays where an enzyme linked to the antibody catalyzes conversion of a chromogenic substrate to a detectable species. Latter are generally more desirable as multiple detectable moieties can be generated per binding event thereby increasing sensitivity. Similar methods have been incorporated into DNA hybridization based assays, which are generally more sensitive and in most diagnostic assays can be used at an earlier stage of disease progression. Enhanced sensitivity is achieved by first amplifying nucleic acid sequence based on the presence of a specific target sequence. Following amplification, the amplified sequences are detected and quantified. As a method of amplifying a nucleic acid sequence, the PCR (polymerase chain reaction) process is known. Presently, PCR is the most conventional means for in vitro amplification of nucleic acid. However, PCR has certain disadvantages, including the requirement for strict temperature control, inadequate quantification due to logarithmic amplification, and the danger of erroneous results brought about by simultaneous amplification of trace amounts of contaminated DNA. Additionally, except for nucleic acids, most other analytes are not easily amplified. In such cases, signal amplification methods are more desirable. These detect amplified decomposition products, i.e., a product or by-product of a reaction is amplified as the signal from a target analyte.
The traditional methods of amplifying the signal use enzyme linked to antibodies or polynucleotides and are limited by the amount of multiplexing one can achieve. There are only a few enzymes, such as alkaline phosphatase or horse radish peroxidase, that have been linked to antibodies or DNA probes. Other methods of signal amplification are based on nucleic acid metabolizing enzymes.
A cycling assay has been developed which utilizes λ-exonuclease to specifically cleave double stranded DNA (C. G. Copley et al., Bio Techniques, Vol. 13, No. 6, pp 882–892, 1992). This method involves hybridizing an oligonucleotide probe with a nucleic acid sequence complimentary thereto, allowing λ-exonuclease to act on the formed double-stranded DNA to decompose the hybridized probe. The probe is replaced by another probe, which is then decomposed. In this way, a cycling reaction repeats. In this method, the presence of a target DNA sequence is estimated by the detection of the decomposed probe. A disadvantage of this method is that the λ-exonuclease requires a probe which is phosphorylated at its 5′-terminal as the substrate. Following chemical synthesis of the probe by known methods, the 5′-terminal needs to be phosphorylated, and it is often difficult to confirm that all 5′-terminals are phosphorylated completely. An additional problem of this method is the low turnover number of cycling reactions, i.e., the number of times hybridization between the primer and target nucleotide occurs. The turnover number is low since the hybridization step must repeatedly occur.
An additional cycling assay by an exonuclease has been disclosed in EP 500224/A1. In this method, the synthesis of a DNA strand complimentary to a target DNA proceeds from a primer simultaneously with the decomposition of the same primer from the other side by a 5′→3′ exonuclease such that another primer hybridizes with the target sequence in place of the decomposed primer hybridized before. Therefore, in a single cycle reaction both the synthesis of a complimentary strand by DNA polymerase as well as the degradation of the synthesized strand repeatedly occurs. A disadvantage of this method is the low turnover number, with the hybridization step being rate limiting in that it must repeatedly occur.
A further cycling assay for detection of a polynucleotide containing a specific sequence is disclosed in U.S. Pat. No. 5,849,487. This method relies on signal amplification and detection of decomposition products. This method includes using a combination of nucleic acid polymerase, 3′→5′ exonuclease, a nuclease-resistant primer, a target nucleic acid, which may be DNA at limiting concentration, and at least one deoxynucleoside triphosphate (dNTP) to detect the target nucleic acid sequence. The method further includes synthesizing a complimentary strand being a nucleotide species located adjacent to the 3′-terminal of the nuclease-resistant primer, followed by decomposition of the nucleotide species joined to the end of the primer and detection of the resulting pyrophosphoric acid or deoxynucleoside monophosphate, the synthesis and decomposition of the nucleotide species being repeated one or more times. A disadvantage of this method as well as other detection methods presently widely in use is the need to separate labeled starting material from a final labeled product or by-product. Such separations generally require gel electrophoresis or immobilization of a target nucleic acid sequence onto a membrane for detection. For example, in U.S. Pat. No. 5,849,487, the deoxynucleoside monophosphate formed by a nuclease reaction is separated by chromotography and optically measured. Alternatively, the pyrophosphoric acid which is formed upon incorporation of a complimentary base by DNA polymerase may be allowed to react with adenosine-5′-phosphosulfate and adenosine triphosphate sulfurase to form adenosine triphosphate, which is then detected using a luciferin-luciferase reaction; this presents the disadvantage of requiring additional reagents and incubation steps.
It has been known that DNA and RNA polymerases are able to recognize and utilize nucleosides with a modification at or in place of the gamma position of the triphosphate moiety. It is further known that the ability of various polymerases to recognize and utilize gamma-modified nucleoside triphosphates appears to vary depending on the moiety attached to the gamma phosphate.
A colorimetric assay for monitoring RNA synthesis from RNA polymerases in the presence of a gamma-phosphate modified nucleotide has been reported (Ref. Vassiliou W, Epp J B, Wang B B, Del Vecchio A M, Widlanski T, Kao C C. Exploiting polymerase promiscuity: A simple colorimetric RNA polymerase assay. Virology. Sep. 1, 2000; 274(2):429–37). In this report, RNA polymerase reactions were performed in the presence of a gamma-modified, alkaline phosphatase resistant nucleoside triphosphate which was modified at its gamma phosphate with a dinitrophenyl group. When RNA polymerase reactions were performed in the presence of this gamma-modified NTP as the sole nucleoside triphosphate and a homopolymeric template, it was found that RNA polymerase could recognize and utilize the modified NTP. Moreover, when the polymerase reactions were performed in the presence of an alkaline phosphatase, which digested the p-nitrophenyl pyrophosphate aldo-product of a phosphoryl transfer to the chromogenic p-nitrophenylate, an increase in absorbance was reported. A disadvantage of this detection method is that the real-time colorimetric assay, performed in the presence of an alkaline phosphatase, only works with a homopolymeric template.
It would, therefore, be of benefit to provide methods of detecting and characterizing an analyte, which methods would include utilization of terminal-phosphate-labeled nucleotides as substrates for DNA polymerase in a cycling assay by an exonuclease. It would further be of benefit if such methods would employ enzyme-activatable labels at the terminal phosphate of the nucleotide for production of an amplified detectable species from a target nucleic acid which would eliminate the need to separate labeled starting materials from labeled products or by-products. Moreover, it would be highly desirable if such methods for detecting and characterizing nucleic acids would allow for real-time monitoring of a heteropolymeric target nucleic acid using routine lab instrumentation. Finally, it is additionally desirable if such methods are easily multiplexed to analyze four or more analytes per reaction compartment simultaneously or in sequential manner.